Novel surfactant stabilized PLGA cisplatin nanoparticles for drug delivery applications

In the current study, cisplatin loaded (Poly(lactic-co-glycolicacid)) (PLGA) stabilized with 4-phenylphenacylbromide based surfactants nanoparticles were developed for drug delivery applications. For the course of this study four new 4-phenylphenacylbromide based compounds abbreviated as PA(C2)3, PA(C8)3, PAC16 and PAC18 have been synthesized by the reaction of 4-phenylphenacylbromide with various long chain amines. The new surfactants were characterized with 1H and 13C NMR and FTIR spectroscopy. The critical micelle concentration (CMC) of newly synthesized surfactants are in the range of 0.024–0.091 mM. The newly synthesized surfactants have been incorporated in PLGA based nanoparticles and are used for drug encapsulation and delivery of cisplatin. The drug encapsulation efficiency for newly fabricated nanoparticles was found to be in the range of 65 ± 1.5 to 67 ± 1.6%, the drug loading was observed in the range of 1.96 ± 0.11 to 2.31 ± 0.19%, whereas the maximum drug release was found to be 85.1 ± 1.1%.

these advantages, the stability of NPs to some extent becomes limited when polymeric NPs are loaded with drugs.One way to increase the NPs colloidal stability is the use of amphiphilic substances called surfactants.Different investigators used range of surfactants from nonionic to ionic (cationic or anionic) [13,14].In a recent study [15] different surfactant, i.e.PVA, Pluronic F68, Pluronic F127 and polysorbates (Tween 20, Tween 80) were used in the preparation of PLGA NPs loaded with protein kinase C inhibitor and the result shows the drug encapsulation efficiency varied from 31 to 75% with a drug loading of 1.3%-2% and partially hydrolyzed PVA was the surfactant of choice.Similarly in another recent study [16], positively charged curcumin nanoparticles were synthesized using PLGA and cationic surfactant cetyltrimethylammonium bromide (CTAB) and was investigated as fungicidal agents.
In the present communication, four new 4-phenylphenacylbromide based surfactants were synthesized by reacting with long chain amines and used as stabilizing the cisplatin loaded PLGA for DD applications.

Experimental 2.1 Chemicals
Chemicals like 4-phenyl phenacylbromide, triethyl amine, trioctyl amine, hexadecyl amine, octadecyl amine, ethylbromide, cisplatin (CP), o-phenylenediamine (OPDA) and solvents like methanol, ethanol, chloroform, acetone, DMSO, DMF, HCl, THF used in this research work were purchased from Sigma Aldrich.Deionized water was used throughout the study.PLGA, cis-platin and o-phenylenediamine used for the drug delivery application were also purchased from Sigma Aldrich.All solutions were freshly prepared and used immediately.

Instrumentation
1 H-NMR and 13 C-NMR analysis of synthesized surfactants were done at 300 MHz using nuclear magnetic resonance (NMR) spectrometer (Bruker) with a 5 mm PROBHD BBO BB-1H probe for 1 H and 13 C NMR at 295 K. Deuterated chloroform (CDCl 3 ) and Dimethyl sulfoxide (DMSO) were used as a solvent.FT-IR spectra of synthesized surfactants were done on BRUKER-TENSOR-27 in the range of 4000 cm -1 to 400 cm -1 (resolution was 1 cm -1 and 15 scan) to get insight about the functional groups and structural composition of surfactants.Melting points were recorded on a capillary tube using electro thermal melting apparatus, model MPD Mitamura (Japan).

Synthesis
The compounds in this series were synthesized by the reaction of 4-phenyl phenacyl bromide using amines such as triethyl amine, trioctyl amine, hexadecyl amine, octadecyl amine.

Synthesis of PA(C 2 ) 3
One gram of 4-phenyl phenacyl bromide was dissolved in 100 mL dry acetone and the resulting solution was transferred to a 250 mL two neck round bottomed flask.The solution was heated up to 70 °C and 0.5 mL of triethylamine was added from a dropping funnel with constant stirring.The reaction mixture was refluxed for 10 h maintaining the reaction conditions.The chemical equation for the reaction is given in Scheme 1.
The resulting brownish precipitates of the cationic surfactant were filtered and washed with ethyl acetate-hexane mixture several times, dried and collected as amorphous brown solid.

Synthesis of PA(C 8 ) 3
One gram of 4-phenyl phenacyl bromide was dissolved in 100 mL dry acetone and the resulting solution was transferred to a 250mL two neck round bottom flask.The solution was heated up to 70 °C and 1.6 mL of trioctylamine was added from a dropping funnel with constant stirring.The reaction mixture was refluxed for 10 h maintaining the reaction conditions.The chemical equation for the reaction is given in Scheme 2.
The brown precipitates of cationic long chain surfactant were filtered and washed with ethyl acetate-hexane mixture, dried and collected.

Synthesis of PAC 16
One gram of 4-phenyl phenacyl bromide and 0.86 g of hexadecyl amine were dissolved in 100 mL dry acetone and the resulting mixture was transferred to a 250mL two neck round bottom flask.The contents were heated up to 70 °C with PROBHD BBO BB-1H probe for 1 H and 13 C NMR at 295 K. Deuterated chloroform (CDCl 3 ) and Dimethyl sulfoxide (DMSO) were used as a solvent.FT-IR spectra of synthesized surfactants were done on BRUKER-TENSOR-27 in the range of 4000 cm -1   to 400 cm -1 (resolution was 1 cm -1 and 15 scan) to get insight about the functional groups and structural composition of surfactants.Melting points were recorded on a capillary tube using electro thermal melting apparatus, model MPD Mitamura (Japan).

Synthesis
The compounds in this series were synthesized by the reaction of 4-phenyl phenacyl bromide using amines such as triethyl amine, trioctyl amine, hexadecyl amine, octadecyl amine.

Synthesis of PA(C 2 ) 3 :
1 g of 4-phenyl phenacyl bromide was dissolved in 100 ml dry acetone and the resulting solution was transferred to a 250 ml two neck round bottomed flask.The solution was heated up to 70 °C and 0.5 ml of triethyl amine was added from a dropping funnel with constant stirring.The reaction mixture was refluxed for 10 hours maintaining the reaction conditions.The chemical equation for the reaction is given in Scheme-1.constant stirring.The reaction mixture was refluxed for 10 h maintaining the reaction conditions.The chemical equation for the reaction is given in Scheme 3.
The white precipitates of neutral compound were filtered and washed with ethyl acetate-hexane mixture, dried and collected and melting point of the product was calculated.

Synthesis of PAC 18
One gram of 4-phenyl phenacyl bromide and 0.97 g of octadecyl amine were dissolved in 100mL dry acetone and the resulting mixture was transferred to a 250mL two neck round bottom flask.The contents were heated up to 70 °C with constant stirring.The reaction mixture was refluxed for 10 h maintaining the reaction conditions.The chemical equation for the reaction is given in Scheme 4.
The neutral product was obtained as white solid and was filtered and washed with ethylacetate-hexane mixture, dried and collected and its melting point was recorded.

CMC calculations
For this study, the CMC of synthesized surfactants has been determined by tensiometric method.0.001, 0.005, 0.010, 0.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.045, 0.050 mM dilutions for each surfactant were prepared from 1 mM stock solution.The value of surface tension for each dilution was measured by placing 30 mL of each dilution in the tensiometer container one by one.These values were then plotted as a function of concentration of the surfactant in the solution and CMC was identified from the graph as the point where the decreasing slope and baseline of minimal surface tension values intersected.

Drug encapsulation in PLGA NPs stabilized with surfactants
The novel surfactants have been used for the drug delivery application of cis-platin, a well-known anticancer drug.The drug is encapsulated with surfactant by nanoprecipitation method, using PLGA as a particle forming agent.The added surfactant from the solution plays a role in stabilization PLGA nanoparticles.PLGA NPs were prepared by a nanoprecipitation method as previously described [17,18] with slight modifications, in brief, 5 mg cis-platin drug and 100 mg PLGA were accurately weighed and added to a test tube.A total of 0.2 mL DMSO and 1.8 mL acetone was added to the test tube and the contents were sonicated for 1 min to get a homogeneous mixture.The resulting organic mixture was added to a 250mL beaker containing 10 mg surfactant solution in 20 mL deionized water under constant stirring.Visible nanoprecipitation was seen to occur.The mixture was stirred for 3 h at room temperature to evaporate organic solvent.Later, the colloidal mixture was centrifuged at 14000 rpm, 1600 g for 15 min to separate nanoparticles from supernatant.The supernatant was decanted, and nanoparticles were washed with deionized water and lyophilized.The set of prepared nanoparticles and their theoretical composition is shown in Table 1.

Spectrophotometric determination of drug encapsulation and release study
The quantification of cisplatin was carried out by a modification of UV-Vis spectrophotometric method (o-phenylenediamine; OPDA-derivatization) previously reported [19].The determination of drug content is based on the The resulting brownish precipitates of the cationic surfactant were filtered and washed with ethyl acetate-hexane mixture several times, dried and collected as amorphous brown solid.

Synthesis of PA(C 8 ) 3 :
1 g of 4-phenyl phenacyl bromide was dissolved in 100 ml dry acetone and the resulting solution was transferred to a 250ml two neck round bottom flask.The solution was heated upto 70 °C and 1.6 ml of trioctylamine was added from a dropping funnel with constant stirring.The reaction mixture was refluxed for 10 hours maintaining the reaction conditions.The chemical equation for the reaction is given in Scheme-2.The brown precipitates of cationic long chain surfactant were filtered and washed with ethyl acetate-hexane mixture, dried and collected.

Synthesis of PAC 16 :
1 g of 4-phenyl phenacyl bromide and 0.86 g of hexadecyl amine were dissolved in 100 ml dry acetone and the resulting mixture was transferred to a 250 ml two neck round bottom flask.The contents were heated upto 70 °C with constant stirring.The reaction with ethyl acetate-hexane mixture several times, dried and collected as amorphous brown solid.

Synthesis of PA(C 8 ) 3 :
1 g of 4-phenyl phenacyl bromide was dissolved in 100 ml dry acetone and the resulting solution was transferred to a 250ml two neck round bottom flask.The solution was heated upto 70 °C and 1.6 ml of trioctylamine was added from a dropping funnel with constant stirring.The reaction mixture was refluxed for 10 hours maintaining the reaction conditions.The chemical equation for the reaction is given in Scheme-2.The brown precipitates of cationic long chain surfactant were filtered and washed with ethyl acetate-hexane mixture, dried and collected.

Synthesis of PAC 16 :
1 g of 4-phenyl phenacyl bromide and 0.86 g of hexadecyl amine were dissolved in 100 ml dry acetone and the resulting mixture was transferred to a 250 ml two neck round bottom flask.The contents were heated upto 70 °C with constant stirring.The reaction mixture was refluxed for 10 hours maintaining the reaction conditions.The chemical equation for the reaction is given in the Scheme-3.The white precipitates of neutral compound were filtered and washed with ethyl acetate-hexane mixture, dried and collected and melting point of the product was calculated.

Synthesis of PAC 18 :
1 g of 4-phenyl phenacyl bromide and 0.97 g of octadecyl amine were dissolved in absorbance measurement of the reaction product of released cis-platin and OPDA.The product was obtained in 10 -5 M HCl at 90 °C in 30 min.The percentage of entrapped cisplatin (drug encapsulation efficiency) was measured by dissolving a dried pellet of known weight with 100 μL of DMF by vortexing for 30 min.For drug release study, the encapsulated cisplatin was released by suspending all sets of prepared nanoparticles in phosphate buffer saline at pH 7.4 one by one for several hours.The mixture was centrifuged at 14000 rpm for 15 min and the resulting supernatant was checked for the presence of cisplatin by UV-Vis absorbance around 710 nm.

Schematic structure of synthesized surfactants
Four new 4-phenyl phenacylbromide based compounds have been synthesized by reacting the precursor with different long chain tertiary and primary amines and three of these compounds are regarded as surfactants.These surfactants are used in the drug delivery application of cis-platin along with PLGA.The structure of 4-phenyl phenacylbromide and the synthesized surfactants are shown in Figures 1 and 2. The physical data of these surfactants are summed up in the Table 2.

Characterization 3.2.1 Characterization by physical parameters
The newly synthesized series can be characterized in terms of physical parameters such as physical state, color, melting point etc., as illustrated in Table 3.All newly synthesized compounds are solids at room temperature and have sharp melting points.All of these shows moderate solubility in common organic solvents as well as in water.

Characterization by spectroscopic data
All new synthesized 4-phenylphencylbromide based compounds have been characterized by 1 H and 13 C NMR spectroscopy and FTIR spectroscopy.The successful synthesis of all 4 surfactants has been confirmed by spectroscopic techniques.

1 H NMR Spectroscopy
The 1 H NMR spectrum was recorded for all 4-phenyl phenacylbromide based compounds.The spectral data is found to be in accordance with the predicted structure hence it can be said that the synthesis of new surfactants was successful.
The 1 H NMR spectrum of PA(C 8 ) 3 is shown in Figure 3.The characteristic peaks and their multiplicity are listed in Table 4. 1

13 C NMR spectroscopy
The number and types of groups of carbon atoms present in the compound are confirmed by 13 C NMR spectroscopy and the predicted structure for the new surfactants can be justified by elaborating the NMR data.The spectrum of PA(C 8 ) 3 is shown in Figure 4.The characteristic shifts are illustrated in  21-32, 22-30.1,22.8-29.7 for long chain carbon, 10.5, 14, 14.5 and 14 for CH 3 , respectively.These characteristic peaks confirm the formation of novel surfactants.
The FTIR data of newly synthesized surfactants is summed up in Table 6.

CMC values
The critical micelle concentration (CMC) i.e. the concentration of a surfactant in a solution at which micelle formation starts, decreases with the increase in chain length of the surfactants.The CMC of newly synthesized 4-phenyl phenacylbromide based surfactants has been determined using tensiometric method.Different dilutions were prepared from 1 mM stock solution of every surfactant and surface tension for each of these dilutions was recorded by a force tensiometer calibrated with distilled water.A graph was plotted between surface tension in nm -1 on y-axis and concentration in mM on x-axis,  and CMC was identified as the intersecting point of two lines, the linear declining slope, and the baseline of minimal surface tension.The CMC plots for the new compounds are displayed in Table 7. Difference in hydrophobic chain length results in different CMC values [1,3].Note that the carbon number in long chain of PA(C 8 ) 3 is the highest (24 carbons in 3 long chains), but the chain length is shorter that is why CMC value is high.Whereas for PAC 16 and PAC 18 surfactants the long chain carbons are lesser (16 and 18 carbons per chain) but the CMC value is lower as compared to PA(C 8 ) 3 surfactant.This makes the PAC 18 surfactant most useful in terms of CMC.

Drug delivery
The drug delivery application of newly synthesized surfactants was carried out using the well-known anticancer drug cisplatin.The drug was encapsulated in PLGA nanoparticles along with surfactants.Surfactants act as encapsulating agents for the drug as well as stabilizers for nanoparticles which were lyophilized and stored for further study.Later the drug release was studied by suspending the resultant nanoparticles in phosphate buffer saline of pH 7.4.

Drug delivery application
The initial CP concentration was taken as 5 mg.The drug encapsulation efficiency can be calculated by determination of encapsulated CP amount by using the following relation.The drug encapsulation efficiency for the prepared surfactants coated PLGA nanoparticle formulations range from 65%-67%, which are very similar to each other.The ease of micelle formation is depicted in the calculated encapsulation efficiency for the formulations prepared using surfactants.The drug loading content was found in the range of 1.96 to 2.31% and very similar for all NPs-surfactant formulations.The results shown above are also comparable to those of the already carried out experiments with cisplatin-PLGA supported with other compounds (mPEG etc) where DLC was found in the range of 1.99%-2.0%[20].In a recent study [21] cisplatin-loaded PLGA NPs supported with chitosan shows the DLC of 6.67 ± 0.9% with an drug encapsulation efficiency of 62.99 ± 2.01%, for human epidermal growth factor receptor 2 targeted ovarian cancer therapy, similarly the other study shows the DLC of 3.9% and DEE of 72% for PLGA-mPEG NPs loaded with cisplatin [22].The release of drug from cisplatin loaded PLGA-surfactant Nps was studied for 96 h and the results shows the release of drug was achieved up to 80.7 to 85.1% which better than the reported previously [22] where nearly 85 % was achieved in 120 h, similarly the other study shows the drug release of less than 40% in 70 h [21].Similarly in another study [23], the percent loading of the PLGA-mPEG nanoparticles with cisplatin was shown to be significant (1%-2.5% w/w).In the proposed method, a modified double emulsion method was used to prepare PLGA-mPEG nanoparticles of cisplatin, which resulted in improved cisplatin loading in the PLGA-mPEG nanoparticles.
Scheme-2Reaction scheme shows the synthesis of PA(C 8 ) 3 Scheme-3Reaction scheme shows the synthesis of PAC 16

Table 1 .
Composition of prepared nanoparticle.

Table 2 .
Description of newly synthesized compounds.

Table 3 .
Physical characteristics of newly synthesized compounds.

Table 4 .
1HNMR data of Newly synthesized compounds.

Table 6 .
FTIR data of newly synthesized compounds.

Table 7 .
CMC values of newly synthesized compounds.

Table 8 .
Drug delivery parameters for cisplatin loaded PLGA-surfactant nanoparticles.

Table 5 .
13C NMR data of newly synthesized compounds.